THE IMPLICATIONS OF GAP JUNCTION INHIBITION IN JURKAT CELL-CELL COMMUNICATION AND PROLIFERATION

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2 THE IMPLICATIONS OF GAP JUNCTION INHIBITION IN JURKAT CELL-CELL COMMUNICATION AND PROLIFERATION A thesis submitted to the Kent State University Honors College in partial fulfillment of the requirements for University Honors by Jeremy Shaw May 2014

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4 Thesis written by Jeremy Shaw Approved by: Co-Advisor Co-Advisor Interim Chair, Department of Biological Sciences Accepted by: Dean, Honors College ii

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6 TABLE OF CONTENTS LIST OF FIGURES...iv ACKNOWLEDGMENT......v CHAPTER I II INTRODUCTION...1 MATERIALS AND METHODS.8 A. Cell Culture and Preparation of Culture Media..8 B. Isolation of CD34+ Cells 8 C. Dye Transfer Assay.9 D. Transwell Assay E. Proliferation Assay 10 F. Real Time Polymerase Chain Reaction. 11 G. Statistical Analysis 11 III RESULTS.. 12 A. Quantifying Gap Junctional Communication in Jurkat Cells B. Gap Junction Communication in Jurkat Cell Requires Direct Cell-Cell Contact..12 C. Dye Transfer Reduced in Presence of Gap Junction Inhibitors D. Jurkat Proliferation Reduced in Presence of Inhibitors 14 E. Heat Map of Connexin expression measured by RT-PCR 15 IV V DISCUSSION 24 REFERENCES..28 iii

7 LIST OF FIGURES Figure 1A: Dye Transfer Assay Procedure Figure 1B: % Calcein Dye Transfer Over 3 Hours Figure 1C: Timepoint-dependent DiI Shift Figure 2: Jurkat Cell Transwell Assay Figure 3A: Carbenoxolone Inhibition of Dye Transfer. 19 Figure 3B: 1-Octanol Inhibition of Dye Transfer.. 20 Figure 4A: Jurkat Proliferation with CBX.21 Figure 4B: Jurkat Proliferation with 1-Octanol. 22 Figure 5: Connexin Expression Heat Map...23 iv

8 Acknowledgements I would like to thank Dr. Justin Lathia s and his colleagues Maksim Sinyuk and Masahiro Hitomi at the Cleveland Clinic for allowing me to learn and perform these experiments as well as for giving me the excellent guidance. I would also like to thank Dr. Douglas Kline for serving as my Faculty Mentor at Kent State for bridging my project to the campus as well as helping to ensure my success. In addition, I would like to thank Case Western Reserve s ENGAGE program and Kent State University s Senior Honors Thesis Fellowship for funding my research. Lastly, thank you to my Thesis Defense Committee members for agreeing to serve on my committee and helping me to establish a strong and complete defense. v

9 1 The Implications of Gap Junction Inhibition on Jurkat Cell-Cell Communication and Proliferation Jeremy Shaw Introduction Due to the relatively short life of mature blood cells in humans, a healthy human body needs to constantly produce new blood cells to replenish its supply. This process of producing new blood cells is known as hematopoiesis. The Hematopoietic Stem Cell (HSC) is the cell at the top of the hierarchy of all blood cells which is responsible for hematopoiesis under normal circumstances. The HSC gives rise to precursors which, in turn, can produce all possible mature cells of different lineages of the blood. These include: megakaryocytes, erythrocytes, lymphocytes, and myeloid cells. In addition to this function, the HSC can also self-renew to produce more HSCs, increasing the ability of these cells to repopulate the blood supply (Orkin and Leonard, 2008). Leukemia is the abnormal formation and growth of cells in the bone marrow that eventually infiltrate the liver, spleen, lymph nodes, and other tissues throughout the body. Leukemia is further characterized by failed differentiation into the more mature blood cells, and overpopulation of the marrow by these naïve blood cell types. The state of leukemic cell maturity and cell type involved are two factors utilized to classify the different types of leukemia. The maturity of the cells, determines whether it is Acute or Chronic, while the cell type involved determines whether it is myelogenous or lymphogenous. Thus, the four major distinctions of leukemia are Chronic Lymphoblastic

10 2 Leukemia, Chronic Myeloid Leukemia, Acute Lymphoblastic Leukemia, and Acute Myeloid Leukemia (Redaelli, et al, 2003). Acute Myeloid Leukemia (AML) is a hematological malignancy which is diagnosed over 14,000 times and subsequently results in over 10,000 deaths per year. Although it can be found in younger patients, AML has a median age of diagnosis at 66 and is characterized by the rapid proliferation and self-renewal of abnormal leukocytes. These leukocytes, in turn, interfere with the body s normal function of hematopoiesis. Patients diagnosed with AML tend to display symptoms such as anemia, decreased number of red blood cells or amount of hemoglobin in the blood, neutropenia, decreased number of neutrophils in the blood, and thrombocytopenia, decreased amount of platelets in the blood. The current standard of care for AML is a chemotherapy regiment in combination with cytarabine/anthracycline followed by stem cell transplantation of autologous, or, more often, allogeneic Hematopoietic Stem Cells (Lowenburg, et al, 1999). Although there has been significant progress made in recent years in elucidating the molecular mechanisms utilized in AML, the disease will still claim the lives of the majority diagnosed with it. The prognosis is slightly more promising for those under the age of 60, however % of patients under 60 years of age can achieve complete remission, but most of these cases result in relapse leaving a 40-50% five year survival rate. In patients older than 60, cure rates diminish to less than 10% and have not significantly changed in over 30 years (Konig and Levis, 2014).

11 3 A number of factors may be responsible for the high mortality rate among individuals with AML. The first of these three main factors is the heterogeneity of the cells. With a large number of different cell types including nearly all lineages of the blood, as well as surrounding stromal cells, it is difficult to pinpoint which cells are causing which effects. Secondly, the fact that these leukemic cells occupy a niche favoring survival and growth, normally utilized in hematopoiesis of HSCs, leads to a major progression of the disease state. Previous studies have also shown that this niche may also be implicated in providing chemotherapy resistance (Lane, et al, 2009). In addition, there is very likely a sub-population of therapeutically-resistant cancer stem cells capable of recapitulating the original tumor and subsequent tumors following most forms of current chemotherapy treatments. (Bonnet and Dick, 1997) In addition to those three major contributors, the hematopoietic microenvironment has also been shown to contribute to the origin of AML as well as its progression. In this compact niche inside of the bone marrow, a heterogeneous population of both hematopoietic stem cells and non-hematopoietic stromal cells can be found. These cells produce a variety of different cytokines, collagens, and other extracellular matrix molecules. Cell to cell communication as well as a cell s communication with its environment is vital in this close-quarters bone marrow niche. These types of communication play a major role in promoting the survival, expansion, and differentiation of Hematopoietic Progenitor Cells in a healthy adult. The same cell-cell interactions and interactions with the extracellular matrix are also important in tumorigenesis and the progression of AML. In recent studies it has been shown that

12 4 leukemic cells directly contacting their surrounding stroma inhibit apoptosis of these cells as well as enhance their survival following chemotherapy treatments (Allouche, et al, 2000). Although evidence has been previously shown to implicate the involvement of exosomal micro RNAs (mirna) as an alternative method of communication between these types of cells, exosomal micro RNAs seem to have less of an effect than direct cell contact communication, and they appear to be more applicable in immune system modulation (Szczepanski, et al, 2011). In addition, there is limited evidence of a mechanism of communication using exosomal RNAs between leukemic cells. The focus of current research, particularly on developing treatments for AML, is directed toward understanding direct cell-cell communication since this has been a previously-unexplored mechanism. Noting the direct contact between leukemic cells and taking into consideration the compact nature of the bone marrow niche, gap junctions are likely to be responsible for direct communication. Gap junctions are defined as cell-cell junctions at which two plasma membranes from contacting cells join each other and share a cytoplasmic bridge. These bridges enable the exchange of small ions, such as Na +, K +, and Ca 2+, and small signaling molecules like camp and inositol 1,4,5 triphosphate (Kumar, et al, 1996). These intercellular connections are specialized and are formed in humans by a family of 21 proteins called connexins. When 6 of these connexins aggregate, they form a connexon on the surface of the cell. As one connexon meets a connexon of a neighboring cell, they form an open pore protected from the extracellular environment allowing for

13 5 direct exchange of small molecules and ions between the cytoplasms of adjoining cells (Sáez, et al,2005). It should be noted, however, that six uniform connexins are not necessary to form the gap junction. Heterogenous combinations of connexins may produce gap junctions that vary in what can be transported, how much is transported, the speed at which it is transported, and other transport regulations (Kumar, et al., 1996) In multiple advanced cancers, gap junctions have been shown to be associated with the invasion, specifically intravasation into the blood vessel, extravasation, and metastasis of tumor cells. These properties promote the progression of the late stages and deadliness of these diseases. In AML, one particular connexin protein, Cx43, is considered a potential tumor promoter which utilizes the exchange of growth factors for tumor progression between cells. Cx43 is also upregulated in regenerating bone marrow; this suggests direct cell-cell communication in the early stages of blood formation in addition to implications in AML (Krenacs, and Rosendaal, 1998). AML is, of course, best studied in primary leukemia cells, however, primary leukemia cells are difficult with which to work. Alternative cells such as Jurkat cells provide a good model system for initial studies. In addition, the outcome of experiments performed with Jurkat cells can help determine whether or not experiments in primary leukemic cells should be conducted. Jurkat cells are an immortalized cell line of human T lymphocytes which was established from the peripheral blood of a 14-year-old boy who suffered from T-cell Leukemia and is used as a model to test for vulnerability of blood cancers to novel methods of treatment.

14 6 The following experiments were performed to determine if gap junctional communication was in fact occurring between Jurkat cells as a model for AML, and what effects gap junctional communication may have on these cells. In these experiments we attempted to elucidate the effects that gap junctional exchanges may have on cell behavior and activity by using compounds that are known to block gap junctional communication. To measure the functional gap junctional activity, dye transfer assays were utilized in the presence of clinically-relevant gap junction inhibitors, Carbenoxolone (CBX) and 1-Octanol. These compounds are able to change the membrane fluidity which prevents the connexins from being able to properly form functional gap junctions (Sáez, et al,2005). Jurkat cell proliferation was also examined in response to these gap-junction inhibitors. Also, to rule out the possibility that communication might be occurring through methods other than through direct cell-cell contact-mediated gap junction communication, a trans-well assay was also performed. This assay utilizes a barrier to physically separate different populations of cells to observe the effects of preventing direct contact on communication. Finally, an experiment was performed to translate the work performed in Jurkat cells to those in a primary AML sample. To discover if particular connexin proteins were being used in gap junction communication in AML as compared to Jurkat cells and normal tissue, a screen was performed of all known connexins which resulted in identifying several potentially targetable connexin proteins. This screen included Leukemic cells, Primary AML cells from a patient as well as Hematopoietic Stem Cells.

15 7 This experiment was particularly important because it was able to establish the baseline for a potential cell-specific targeting of AML cells without a multitude of off-target effects which may have been caused by a ubiquitous blocking of all gap junctions in all cell types.

16 8 Materials and Methods Cell Culture and Preparation of Culture Media For the majority of the experiments, Jurkat cells were used. In the final experiment involving specific connexin identification, however, primary patient AML samples were used. The Jurkat cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, (Dr. Huang, Cleveland, USA), 2mM sodium pyruvate, 5x10^-5 M 2-Mercaptoethanol, penicillin, streptomycin and.1mm nonessential amino acids at 37 C in a humidified atmosphere of 20% oxygen and 5% CO2. The Primary AML were obtained from cord blood or bone marrow and were cultured in IMDM supplemented with 10% fetal bovine serum and 10ng/ml of the following human cytokines: stem cell factor (SCF), FLT3 ligand, thrombopoietin, interleukin-3 (IL-3) and interleukin-6 (IL-6). Isolation of CD34+ Cells CD34+ cells were obtained from bone marrow aspirates and were immunopositively purified using a magnetic sort system. (CD34 MicroBead Kit, #: , Miltenyi Biotec, Auburn, CA, USA) following the manufacturer s instructions. These normal hematopoietic stem cells were subsequently cultured using the same media as primary AML cells mentioned in the previous methods.

17 9 Dye Transfer Assay This experiment utilizes the properties of two dyes, Calcein AM and DiI to accurately visualize and quantify the type of exchange occurring between cells. To begin, the Jurkat Cells were divided into two groups. The first group was labeled with Calcein AM (Life Technologies). Calcein AM is a cell-permeable dye in its parent form and is converted to a green-fluorescent form of Calcein AM after acetoxymethyl ester hydrolysis by intracellular esterases. At this point, it is no longer cell-permeable and is only able to leave the cell through gap junctions or exocytosis. The other population of cells was labeled with DiI (Life Technologies). This dye is a lipophilic membrane stain that diffuses laterally to stain the entire cell and does not leave the membrane for the life of the cell. Knowing only Calcein AM can be transferred through gap junctions while DiI stays fixed in membranes allows for the dyes to serve their different purposes. The amount of Calcein AM transferred is representative of the amount of gap junction communication occurring while the DiI stain serves as a background stain which should remain unaffected throughout the experiment despite incubation periods or conditions. After loading the two populations of cells, the Calcein AM and DiI-labeled cells were mixed at a 1:1 ratio and incubated together for 0.5, 1, 2, and 3 hours in a 15mL conical tube at 37 C. After the designated time period had passed, the cells were analyzed using flow cytometry to quantify the amount of dye transfer which occurred. After this initial experiment had been performed, it was repeated for the same time points in the presence of different concentrations of one of two gap junctional inhibitors.

18 10 Carbenoxolone (CBX) and 1-Octanol. CBX concentrations were 1µM, 50 µm, and 500µM while 1-Octanol was used at concentrations of 100 µm, 500µM, and 1mM. Transwell Assay Two separate populations of cells were labeled with Calcein AM and DiI dyes. Rather than mixing these cells together, they were incubated on opposite sides of Transwell inserts with a 0.4µm pore (Corning, USA) for periods of 1, 2, & 3 hours. These Transwells served to effectively physically separate the two populations of cells in culture. Following the incubation periods, dye transfer was measured using flow cytometry. Proliferation Assay To measure leukemic cell proliferation in the presence of the gap junctional inhibitors CBX and 1-Octanol, a CellTiter-Glo proliferation assay (Promega, USA) was used. The assay measures the number of cells in the sample by quantifying the amount of ATP present. This is an effective method in a relatively homogenous population of cells which works by detecting cells with active metabolisms. One thousand cells were placed in clear-bottom 96-well plates with 100µL of the culture media. At least two hours passed before the first day 0 measurement was taken. On days 0, 1, 3, 5, and 7, luminescent readings were obtained. This was done by adding 100µL of CellTitler-Glo reagent to each of the wells and incubating the cells on a rocker protected from the light for a period of 30 minutes; the luminescence was then measured using a Victor 3 luminometer. To

19 11 account for any variation during preparation of the cells, all of the data used to generate relative growth curves was normalized to day 0. Real Time Polymerase Chain Reaction Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to manufacturer s instructions. Complimentary DNA (cdna) synthesis and subsequent amplification from 1 µg messenger RNA (mrna) was performed using the qscript cdna Supermix (Quanta Biosciences, USA) with an Eppendorf Vapo. Protect Mastercycler Pro (Eppendorf USA). Gene expression was measured by Quantitative Real-Time PCR (qpcr) using the RT 2 SYBR Green ROX qpcr Mastermix Kit (SABiosciences, USA). Custom Primers were created for each of the 20 different connexins. The PCR reaction and detection were performed with the ABI 7000 Sequence Detection System (Applied Biosystems, USA). Connexin levels were calculated and normalized to B-actin and GAPDH. Statistical Analysis Data are reported as mean values with standard deviation. Statistical significance was analyzed using one-way ANOVA. P values less than 0.05 were considered to be significant.

20 12 Results Quantifying Gap Junctional Communication in Jurkat Cells A dye transfer assay was first utilized to assess whether gap junctional communication was present and detectable in Jurkat cells. The process as outlined in the methods section is diagrammed in Figure 1A. Groups of cells were labeled with DiI or Calcein and then mixed and incubated together. Flow cytometry analysis indicated a 3.7±0.42% level of fluorescence of Calcein transfer as seen in Figure 1B which confirms that connexin channels are functional and active Jurkat cells. Furthermore, increasing the amount of time in which the mixed population of cells is allowed to incubate allows for a greater amount of cell-cell contact and more dye transfer through gap junctions to occur. After one hour of incubation, the rate of transfer had increased to 14±1.5%, after another hour had passed, the percentage of transfer increased to 73±2.8%, and at the final 3-hour time point, the rate had increased to 90±3.6%. The rate of Calcein transfer over time was also validated by measuring the percent of fluorescence intensity over time in Figure 1C. The data indicates that as more cells are allowed to interact with each other through gap junctions, Calcein is being transferred in greater amounts which in turn amplifies the intensity of the following fluorescent signal. Gap Junction Communication in Jurkat Cell Requires Direct Cell-Cell Contact To demonstrate that physical contact is necessary for transfer, thus decreasing the probability of exosomal communication mechanisms, a Transwell assay was designed

21 13 and performed. After 1 and 3 hour time points of incubation, the cells which were allowed normal contact had Calcein transfer rates of 7.7% and 24%, while the cells incubated in Transwells exhibited far reduced transfer rates of 1.2% and 1.3%. Although some double-positive results were observed, cells which had large amounts of both Calcein and DiI, this is likely because this experiment does still allow for exosomal transport to occur. If exosomal transfer were occurring, that would use part of the DiI-stained membrane to transfer the exosome to the Calcein AM-stained cells, causing cells to have large amounts of both dyes. Although previous work has suggested exosomal communication occurs between leukemic cells and endothelial cells, this result suggests that little or no exosomal transport is occurring in these cells under the conditions of these experiments. Since the amount of Calcein AM transferred is still minimal with the separated cells, this experiment also discounts the theory of Calcein AM uptake by the cells from the culture media. Thus, Calcein AM transfer is dependent on direct cell-cell contact and transfer through gap junctions. Dye Transfer Reduced in Presence of Gap Junction Inhibitors Since transfer of Calcein had already been shown, the next step was to view the effects Gap Junction inhibitors would have on this process. Two FDA-approved compounds, Carbenoxolone (CBX) and 1-Octanol were used as gap junction inhibitors in the Jurkat cells. Similar to the previous experiment, two populations of cells were stained with Calcein and DiI. However, they were also incubated in the presence of one of two

22 14 concentrations of two inhibitors before being mixed for a period of 1 or 3 hours. After this incubation period, the amount of Calcein transfer was to be measured. In the control group with no inhibitors, transfer rates of 5.1% and 45% were observed. At the 1µM concentration of CBX however, dye transfer was 0.57% at one hour and 1.9% at three hours, and in the 100µM group, transfer rates of 0.61% and 4.6% respectively were observed. In the 50µM concentration of 1-Octanol, there was slightly increased 8.8% transfer at one hour, but a reduced 29% transfer at three hours when compared to the control group in both instances. Though, at the higher 1mM concentration of 1-Octanol, transfer only occurred at 1.15% at one hour and 3.73% at three hours. All of this data, as seen in Figure 2, confirm that in-vitro gap junction activity can be blocked in a dose-dependent manner by CBX as well as 1-Octanol. Jurkat Proliferation Reduced in Presence of Inhibitors The proliferation of leukemic cells is central to the progression of AML. Treatment of the disease may depend on developing methods to inhibit the proliferation of these cells. Since gap junctional communication is associated with proliferation it would be useful to know if introduction of gap junction inhibitors could reduce cell proliferation. Jurkat cell proliferation was examined in groups of cells treated with both CBX and 1-Octanol. Both inhibitors decreased Jurkat cell proliferation. At the 50µM and 100µM concentrations of CBX, a 1.8 and 190-fold reduction in cell proliferation occurred in the treated cells. Similarly, Jurkat cells showed a 1.2-fold reduction in growth

23 15 and proliferation when exposed to the 100µM concentration of 1-Octanol and a 2.0-fold reduction at the 500µM concentration. Heat Map of Connexin Expression Measured by RT-PCR To discover if specific connexins were involved in gap junction communication in Jurkat as well as Primary AML cells, an RT-PCR screen of all known connexin mrna was employed. Normal HSCs were used as a baseline control to emphasize which connexins were upregulated in the Jurkat as well as AML primary cells. In this screen, four connexins were found to be upregulated in Jurkat cells and in a primary AML specimen: Cx25, Cx31.9, Cx40, and Cx62 (Figure 5).

24 16 Fig 1: Dye Transfer Experiment (A) is a schematic detailing the uptake of the two different dyes Calcein AM and DiI into two separate populations of cells, the wash steps, and the transfer of the Calcein AM dye to the DiI-labeled population of cells during mixed incubation.

25 17 (B) shows the percent of Calcein transferred between Jurkat cells over time during mixed incubation. (C) displays the number of cells which displayed 100% of Calcein transfer after the different lengths of time incubating according to fluorescence readings.

26 18 Fig 2: Transwell Experiment DiI is represented by the purple population of cells while the Calcein is represented by the green population. The prevalence of cells from the top far left of the graph compared to the top mid left section being examined is showing the transfer of Calcein to cells which previously only contained DiI; the mechanism for this transfer is gap junctions. The amount of transfer in the top two control graphs is far greater than the amount of transfer in the bottom two graphs which utilized Transwells.

27 19 Figure 3A Carbenoxolone Inhibition on Dye Transfer In the first column, the control group, or cells incubated without Carbenoxolone, a large amount of Calcein transfer can be seen occurring at both hour one and hour three. However, in the presence of 1µM as well as 100µM concentrations of Carbenoxolone, a great reduction in Calcein transfer can be seen in the mixed populations. This not only occurs at the one hour time point, but at the three hour one as well.

28 20 Figure 3B 1-Octanol Inhibition on Dye Transfer The first column, the control group, is the same as in the last figure. After one hour in the presence of 50µM concentration of 1-Octanol, the amount of Calcein dye was decreased by only minor amounts compared to the control with no inhibitor. When the concentration of 1-Octanol is increased to 1mM, however, there is a steep decline in the amount of Calcein being transferred. Both the inability of the 50µM concentration to effectively block the dye transfer as well as the success of the 1mM concentration remain valid when moving to the three hour time points as well.

29 21 Figure 4A: Jurkat Cell Proliferation with Carbenoxolone Jurkat cells in the control group, the group without any CBX, maintained a steadily increasing proliferative capability. However, when these same cells were grown in the presence of 50µM CBX, a marked and significant decrease in proliferation could be observed. At the most drastic level, when CBX s concentration was increased to 100µM, a massive decreased in proliferation could be observed. This data implies a dosedependent effect on Jurkat cell proliferation.

30 22 Fig 4B: Proliferation in the Presence of 1-Octanol Bearing a similar trend to the previous figure, the Jurkat Cell control group proliferated in a rapid fashion in the absence of 1-Octanol. When a 100µM concentration of 1-Octanol was used, however, proliferation showed a noticeable increase. Although not quite as grand of a proliferative decrease occurred in the 500µM concentration of 1-Octanol as Carbenoxolone s data, there is still a large decrease in Jurkat cell proliferation which follows a concentration-dependent trend.

31 23 Figure 5: Heat Map of Connexin Expression This figure is displaying the expression of connexin mrnas with regards to relative expression of those same connexins found in Hematopoietic Stem Cells (HSCs). A red color signifies a high expression in AML and a low expression in HSCs, while a blue color signifies a low expression in AML and a high expression in HSCs. Accordingly, white backgrounds mean a similar amount of expression in AML as well as HSCs. Since this screen is searching for AML-specific targeting mechanisms which won t affect HSCs, the connexins with double red on the heat map will pose the greatest potential for further studies.

32 24 Discussion Through these experiments, we have established that gap junctional communication occurs in Jurkat cells, and that this communication can be blocked by the compounds CBX and 1-Octanol. In addition, the presence of these inhibitors has shown a marked decrease on the proliferation of the Jurkat cells which leads to the conclusion that inhibiting communication in these cells has adverse effects on their ability to proliferate. Furthermore, from the Transwell experiment, it has been established that cytoplasmic exchange through gap junctions is reliant on direct physical contact between cells. Finally, the identification of specific primers using the RT-PCR is a vital first step in identifying possible therapeutic targets without the off-target effects which may result in new effective therapies. Although it has been previously shown that heterotypic communication between leukemic cells and their surrounding stromal cells may have positive effects relating to survival and chemotherapy resistance, the results established here suggest a new and unexplored ideology. Homotypic communication, or communication between cells of the same type, between Leukemic cells through gap junctions has the potential to effect the malignancy of AML. Populations of cells are better able to respond to external stimuli and escape damage from sources such as chemotherapy and radiation through the exchange of information amongst themselves. As opposed to simple communication, it also should be considered that gap junctions have strong effects on other roles in the cell. It has already been established that proliferation is impacted by blocking these junctions, but other roles which need careful

33 25 regulation such as differentiation, cell cycle progression, and cell death may also be affected. As one noteworthy possibility, these gap junctions may allow for the release or diffusion of potentially-lethal intracellular components, such as reactive oxygen species, (ROS), or even the uptake of other molecules with may protect them from the damaged to be caused by these ROS. If this is, in fact, occurring, through the more effective blockage of these gap junctions, the ROS may be trapped inside the cells of interest causing a method of cell death in this sub-population of cells promoting the disease. Though these specific connexin proteins show promise in treating AML, their ablation is not enough to specifically target and destroy AML cells. Although they do not share upregulation with HSCs in the bone marrow, it does not necessarily follow that there will not be off-target effects in other cells in the marrow, nor does it discount the possibility of upregulation of different connexin proteins as a method of adaptation in leukemic cells in response to the treatment. However, this method of connexin targeting may serve to be an effective component of an adjuvant form of treatment which combines chemotherapy and radiation. Through this type of therapy, specific AML targeting could occur without affecting the normal hematopoiesis of HSCs. This may be due to the strong non-covalent links between docked connexons which promote adhesive properties for the cell. The bone marrow s high volume of cells in a small area exemplifies its potential as a targetable region to affect cell-cell communication, and appears to be the best route to deliver gap junction-inhibiting drugs in conjugation with current standard of care methods.

34 26 This certainly does not mark the end of the experiments to be performed either. The first of many future experiments to be performed is to repeat all of the above experiments using the primary AML cells. Although Jurkat cells do serve as an effective baseline cell type for showing potential effects in primary AML cells, it is still necessary to repeat these experiments using those cells to note any major differences before moving into future experiments. The proliferation assay could also be repeated with an added control of using HSCs in an effort to further understand the effects that CBX and 1-Octanol may have on regular hematopoiesis. Whether the HSCs, and subsequently the hematopoietic process in the body, is altered in a negative or positive manner, then this could determine whether or not simple broad-spectrum gap junction inhibition could serve as an effective additive treatment in humans currently suffering from AML. Also, although both 1-Octanol and CBX have been shown to have strong, concentration-dependent effects on blocking gap junctions separately, it would be beneficial to determine the effects of using the two in conjugation. It is possible that a deleterious or competitive effect may occur when both compounds are altering the membrane fluidity, but it is also likely that the two compounds may have an additive effect when used together. This could be further explored by finding the best concentration of each of the compounds for this tandem experiment. Although the effects on proliferation have been observed, there are still many cell functions which have yet to have been explored in relation to gap junction inhibition. For example, targeting connexins may also influence leukemic cell adhesion and migration,

35 27 both of which play vital roles in cancer. Experiments which test these cellular functions as well as those such as cell-cycle progression, and escape from apoptosis would also serve to vastly increase our knowledge about how gap junctions work and which vital cellular processes are altered when gap junctions are inhibited. Finally, a knockdown mouse model could be utilized. By using short hairpin RNAs (shrnas) of particular connexin proteins in a mouse model of AML, one would be able to potentially reduce the expression of these connexin proteins and note the differences observed in cell behavior. Since this would be an in-vivo model as well, it would also allow for the observation of implications of some other cell functions such as adhesion and migration in addition to cell-cell communication.

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37 29 9. Redaelli, A., Stephens, J. M., Laskin, B. L., Pashos, C. L., & Botteman, M. F. (2003). The burden and outcomes associated with four leukemias: AML, ALL, CLL and CML. Expert review of anticancer therapy, 3(3), Sáez, J. C., Retamal, M. A., Basilio, D., Bukauskas, F. F., & Bennett, M. V. (2005). Connexin-based gap junction hemichannels: gating mechanisms. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1711(2), Szczepanski, M. J., Szajnik, M., Welsh, A., Whiteside, T. L., & Boyiadzis, M. (2011). Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-β1. haematologica, 96(9),